Method of screening cell death modulators

Abstract

The invention provides methods and compositions for identifying agents which modulate cell death, indicated e.g. by the expression of caspase-2 and/or caspase-7, in GDNF family growth factor deprived neuronal or nonneuronal cells. The methods for identifying such agents find particular application in drug development.

1. A method of screening caspase-2 and caspase-7-dependent cell death pathway modulators of neuronal cells in a glail cell line-derived neurotrophic factor (GDNF)-dependent cell culture system, comprising the steps of:
 removing GDNF from the culture system; 
 introducing a candidate modulator agent into the culture system; 
 determining the activity of at least caspase-2, caspase-3 and caspase-7 in a cultured neuronal cell from said culture system; and 
 identifying said candidate modulator agent as a stimulator of caspase-2 and caspase-7-dependent cell death pathway, if the activity of caspase-2 and caspase-7, and/or the amount of caspase-2 and caspase-7 mRNA or protein is detected to be higher in the presence of the candidate modulator agent than in its absence and caspase-3 is determined to be not activated by the presence of the candidate modulator agent, or identifying said candidate modulator agent as an inhibitor of caspase-2 and caspase-7-dependent cell death pathway, if the activity of caspase-2 and caspase-7 and/or the amount of caspase-2 and caspase-7 mRNA or protein is detected to be lower in the presence of the candidate modulator agent than in its absence and caspase-3 is determined to be not activated by the presence of the candidate modulator agent. 
 
     
     
       2. The method according to  claim 1 , wherein said neuronal cells are selected from the group consisting of cervical ganglion neurons, dorsal root ganglion cells, nodose ganglion neurons, spinal motoneurons, midbrain dopaminergic neurons, central noradrenergic neurons and enteric neurons. 
     
     
       3. The method according to  claim 1 , wherein said candidate modulator agent is from a combinatorial library selected from the group consisting of: biological library; peptoid library, spatially addressable parallel solid phase library, solution phase library; synthetic library requiring deconvolution; “one-bead, one-compound” library; and synthetic library using affinity chromatography selection. 
     
     
       4. The method according to  claim 3 , wherein said combinatorial library is peptide library, non-peptide oligomer library or small molecule library. 
     
     
       5. The method according to  claim 1 , wherein the determining of the ability of the candidate modulator agent to modulate caspase-2 and caspase-7 activity comprises monitoring cell death, cell growth, cell attachment, neurite outgrowth, and/or cell chemotaxis. 
     
     
       6. The method according to  claim 1 , wherein the determining of the ability of the candidate modulator agent to modulate caspase-2 and caspase-7 activity comprises Western blot; immunohistochemical staining using anti caspase-2, caspase-3 or caspase-7 antibodies; and/or fluorometric assays. 
     
     
       7. The method according to  claim 6 , wherein said fluorometric assay is a fluorescence electron transfer (FRET)-assay. 
     
     
       8. The method according to  claim 1 , wherein the determining of the ability of the candidate modulator agent to modulate caspase-2 and caspase-7 activity comprises detecting catalytic or enzymatic activity of caspase-2, caspase-3 and caspase-7. 
     
     
       9. The method according to  claim 1 , wherein the determining of the ability of the candidate modulator agent to modulate caspase-2 and caspase-7 activity comprises detecting a target-regulated cellular response selected from the group consisting of cell attachment, cell adhesion, cell growth, cell death and cell migration. 
     
     
       10. The method according to  claim 1 , wherein the determining of the ability of the candidate modulator agent to modulate caspase-2 and caspase-7 activity comprises detecting the amount of caspase-2, caspase-3 and caspase-7 mRNA or protein in a cultured neuronal cell in the presence of the candidate modulator agent and comparing the detected amount to the amount of caspase-2, caspase-3 and caspase-7mRNA or protein in the cultured cell in the absence of the candidate modulator agent. 
     
     
       11. The method according to  claim 1 , wherein the identification of said candidate modulator agent to stimulate cell death in a GDNF dependent cell culture system is further confirmed by the detection of the presence of activity of caspase-2 and caspase-7 and the absence of activity of Bax, caspase-3, caspase-8, and caspase-9.